As engraftment of patients after bone marrow transplant (BMT) is the only true test for the existence of the pluripotent hematopoietic stem cell (PHSC) and experimental BMT in humans is ethically impossible, In vitro correlates have been sought to identify and characterize the function of the PHSC. We and others have grown CFU-Blast cells from human hematopoietic tissue; however.. our experience with this in vitro colony forming cell has led us to conclude that this cell may not pass the engraftment test due to its limited-self-renewal potential (an important characteristic of the PHSC). Long-term marrow culture (LTMC) for human PHSC as currently performed also remains wanting as the culture system usually cannot be maintained for more than 2 months. Recently a mutant mouse has been suggested to provide the correct microenvironment for human PHSC to grow and differentiate. We have done extensive testing of this model and have found that while human marrow appears to reside in the marrow space of the murine host, absolutely no proliferation of hematopoietic progenitors of human genotype have been observed from 4 weeks to 6 months following transplant into these mice. It therefore becomes essential to search for new approaches to assay the fractions containing PHSC isolated from the human bone marrow. We have drawn heavily from our experience in mice which allowed us to experimentally transplant our fractionated populations. We believe the parallels between the experimental model (mice) and humans will permit us to isolate the human PHSC. We will develop a short-term self-renewal assay using limiting numbers of purified PHSC to determine the ability of these cells to replate in clonogenic assay. We will also develop LTMC assays modified to allow extended proliferation and differentiation of PHSC in vitro. Finally, we will begin clinical phase I trials to examine the effect of transplanting PHSC on the incidence of graft failure in allogeneic BMT.